Perennial crops such as sugarcane, switchgrass, Miscanthus and woody species are major sources of carbon fixed in the form of simple sugars or complexes mixtures of cellulose and hemicellulose. These biomass resources are major targets for the several industries, such as the bioenergy industry, that are currently focused on developing resources demanded by the increasing world population.
Biomass resources are useful, for example, for the production of cellulosic ethanol that could potentially displace 30% of USA current petroleum consumption in the near future. Perlack et al., BIOMASS AS FEEDSTOCKS FOR A BIOENERGY AND BIOPRODUCTS INDUSTRY: THE TECHNICAL FEASIBILITY OF A BILLION-TON ANNUAL SUPPLY, ORNL/TM-2005/66 (2005). These lignocellulosic feedstocks have been proposed to offer environmental and economic advantages over current energy resources, because they require fewer agricultural inputs than annual crops and can be grown on agriculturally marginal lands. Hill et al., Proc. Natl. Acad. Sci. USA, 103: 11206-210 (2006).
Projections have been made showing that the world demand for wood is expected to grow by 20% in the next decade, due to an increasing usage of forest products, woody residues, and woody energy crops for electricity, fuel and biomaterial production. Strauss and Bradshaw, TREE BIOTECHNOLOGY IN THE NEW MILLENNIUM: INTERNATIONAL SYMPOSIUM ON ECOLOGICAL AND SOCIETAL ASPECT OF TRANSGENIC PLANTATIONS, Oregon State University (2001); Mead, Biomass and Bioenergy, 28: 249-66 (2005).
Therefore there is a need to develop highly productive tree plantations to reduce the pressure on natural forests, preceded by extensive breeding advances in plantation tree species such as poplar and eucalyptus. Until now this has been difficult because the long generation time in trees makes conventional breeding a very slow process. Genetic engineering techniques have the potential to greatly shorten the breeding timeline for trees and allow for more targeted breeding.
Approaches to increase carbon allocation to the above ground portions of plants would increase growth rates and biomass yields. Ragauskas et al., Science, 311: 484-89 (2006). In trees the fixed carbon is accumulated mainly in the secondary walls of the cells, which are the major constituent of wood. Secondary walls are composed mainly of cellulose, hemicelluloses and lignin. During secondary wall formation, the biosynthesis of these cell wall components is highly coordinated and depends of master regulatory genes controlling a huge array of individual genes. Despite the advances in the study of secondary wall biosynthetic genes, little is known about the molecular mechanisms underlying the coordinated expression of these genes during wood formation. Zhong et al., Plant Cell, 19: 2776-92 (2006).
There are several types of regulatory processes controlling gene expression, protein production, and protein processing and protein activity. One of such processes involves the activity of transcription factors, which are proteins capable of recognizing sequences in the promoter of genes and, by binding in such particular sequences, modulate the transcription rate of such genes. Several transcription factors have been identified in a number of organisms and their role in controlling particular biosynthetic pathways has been established. For example, transcriptional profiling of genes differentially expressed during in vitro xylem differentiation in Zinnia (Demura et al., Proc. Natl. Acad. Sci. USA, 99: 15794-99, 2002) and Arabidopsis (Kubo et al., Genes Dev., 19: 1855-60, 2005) or during secondary growth in Arabidopsis stems and roots (Oh et al., J. Exp. Bot., 54: 2709-22, 2003; Zhao et al., Plant Physiol., 138: 803-18, 2005) and poplar (Hertzberg et al., Proc. Natl. Acad. Sci. USA, 98: 14732-137, 2001) has led to the identification of diverse families of transcription factors, which may be involved in the regulation of xylem differentiation or secondary growth. Similarly, microarray analysis showed that 182 transcription factors are differentially expressed during different developmental stages of Arabidopsis inflorescence stems. Ehlting et al., Plant J., 42: 618-40 (2005). Although the exact functions of most of these xylem- or secondary growth-associated transcription factors are unknown, they provide useful tools to dissect the molecular mechanisms controlling the complex process of xylem development, including the initiation of differentiation, cell elongation and secondary wall thickening.
Among these xylem- or secondary growth-associated transcription factors are a group of DOF (for DNA-binding with One Finger) domain transcription factors. DOF proteins are plant-specific transcription factors that share a highly conserved N-terminal DNA-binding domain and a C-terminal domain for transcriptional regulation. Yanagisawa, Trends Plant Sci., 7: 555-60 (2002). The DNA-binding domain is characterized by 52 amino acid residues structured as a Cys2/Cys2 Zn2+ finger, which recognizes cis-regulatory elements containing the common core 5′-AAAG-3′ (SEQ ID NO: 5). Umemura et al., Plant J., 37: 741-49 (2004); Yanagisawa & Schmidt, Plant J., 17: 209-14 (1999).
DOF proteins have been suggested to participate in the regulation of biological processes exclusive to plants such as light-regulated gene expression, photosynthetic carbon assimilation, accumulation of seed-storage proteins, germination, response to phytohormones, guard cell-specific gene expression, flavonoid metabolism and lipid biosynthesis. Plesch et al., Plant J., 28: 455-64 (2001); Moreno-Risueno et al., Plant J., 51: 352-65 (2007); Wang et al., Plant J., 52: 716-29 (2007).
In rice the most presented cis element for all seed-preferential transcriptional factor genes was found to be ‘AAAG’ (SEQ ID NO: 5), the core site required for binding of D of proteins, suggesting an essential and most remarkable role of DOF transcription factors in hierarchical regulatory networks controlling rice seed development. Duan et al., Plant Mol. Biol., 57: 785-804 (2005).
Maize DOF1 expresses in leaves, stems and roots and has different transactivation activities in greening and etiolated protoplasts. DOF1 is activated in illuminated leaf cells and may be involved in the light regulation of genes coupled to light-dependent processes. Yanagisawa and Sheen, Plant Cell, 10: 75-90 (1998). Maize DOF1 also has been suggested to be a regulator for C4 photosynthetic phosphoenolpyruvate carboxylase, which catalyzes the primary fixation of CO2 in the C4 photosynthetic pathway. Additionally, it enhances transcription from the promoter of a cytosolic orthophosphate dikinase. Both enzymes are involved in amino acid synthesis and the recapture of respired CO2. It has been proposed that maize DOF1 might play a more general role in the expression of multiple genes related to carbon metabolism. See Yanagisawa, Plant J., 21: 281-88 (2000).
Another maize endosperm-specific DOF protein, named prolamin-box binding factor (PBF), was shown to interact with the basic leucine zipper protein Opaque2, a known transcriptional activator of prolamin gene expression (Vicente-Carbajosa et al., Proc. Natl. Acad. Sci. USA, 94: 7685-90, 1997). Other homologous proteins exist in the endosperm of other cereals, such as BPBF (barley PBF) and WPBF (wheat PBF), both with similar DNA-binding properties as maize PBF. These observations suggest an evolutionary conservation of the PBF gene function, as an important regulator of storage protein gene expression among small grain cereals, and support a scenario where protein-protein interactions are important in the DOF functions. Mena et al., Plant J., 16: 53-62 (1998).
In rice there is a member of the DOF family (OSDOF3) that has been shown to interact with a R2R3-type MYB transcription factor in the aleurone layer, resulting in the induced expression of a number of genes encoding hydrolytic enzymes (α-amylases and β-glucanases) that participate in the mobilization of stored molecules. Washio, Plant Physiol., 133: 850-63 (2003). Gene regulation in these aleurone cells is under the control of phytohormones, mainly the ratio of gibberellins (GA) to abscisic acid (ABA). The observed accumulation pattern of the barley PBF transcript upon seed imbibition suggested that it may be up-regulated by GA and function as a transcriptional repressor upon germination through interaction with the pyrimidine box of the GARC (GA responsive complex), a conserved cis-element required for GA induction identified in hydrolase genes from cereals. Mena et al., Plant Physiol., 130: 111-19 (2002).
In Arabidopsis there are 36 members of the DOF family, two of which, DAG1 and DAG2 (Dof Affecting Germination), also affect seed germination by light response and gibberellin concentration, possibly playing opposite regulatory roles on the same maternal gene(s). Gualberti et al., Plant Cell, 14: 1253-63 (2002).
Additionally, DOF proteins have been described as part of a regulatory network controlling secondary metabolites. In this regard, OBP2, a DOF gene prominently expressed in the phloem of leaves and other organs in Arabidopsis, has been shown to activate expression of CYP83B1, a gene that participates on the synthesis of glucosinolates, a group of secondary metabolites that function as defense substances against herbivores and micro-organisms. Skirycz et al., Plant J., 47: 10-24 (2006). Another Arabidopsis DOF gene member, AtDOF4;2, was identified as a gene inducing a bushy plant phenotype and potentially being involved in the regulation of phenylpropanoid metabolism.
Constitutive overexpression and RNAi-mediated silencing of AtDOF4;2 caused reciprocal changes in the expression of flavonoid biosynthetic genes and the accumulation of flavonoids under cold and high-light conditions. See Skirycz et al., New Phytol., 175: 425-38 (2007).
The participation of DOF proteins in the regulation of phenylpropanoid metabolism has also been shown in Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1). These mutants deposit lignins in cells where these polymers would not normally be found. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and DOF transcription factor families may contribute to the ectopic lignifications phenotypes in these mutants. Rogers et al., New Phytol., 168: 123-40 (2005).
In poplar there are 41 DOF genes, according to Yang et al., Plant Physiol., 142: 820-30 (2006). A sequence analysis of these genes along with 36 Arabadopsis and 30 rice DOF genes revealed 41 conserved motifs, of which one:
(SEQ ID NO: 6)EILKCPRCDSMNTKFCYYNNYNLSQPRHFCKTCRRYWTKGGALRNVP VGGGCRKNKR,was identified as the DOF domain. Id., page 824 and Table I. A maximum-likelihood phylogenetic tree, constructed using full-length protein sequences of these DOF genes, also highlighted 27 pairs of paralogous genes in the terminal nodes of the tree. Id., page 825 and FIG. 2.